Genetic variation in Austrostrongylus thylogale Johnston & Mawson, 1940 (Nematoda: Trichostrongylida) from the tammar wallaby, Notamacropus eugenii (Gray), and the quokka, Setonix brachyurus (Quoy & Gaimard) (Marsupialia: Macropodidae) in Australia.

BACKGROUND: Australian marsupials harbour a diverse array of helminth parasites. Despite current attempts to assess the extent of this diversity in macropodid hosts, it has been suggested that unique parasite fauna of Australian wildlife is difficult to document comprehensively due to the common occurrence of cryptic species. This paper assessed genetic variation within Austrostrongylus thylogale Johnston & Mawson, 1940 from the tammar wallaby, Notamacropus eugenii (Gray), and the quokka, Setonix brachyurus (Quoy & Gaimard), from different localities using the molecular characterisation of the internal transcribed spacers (ITS) within the nuclear ribosomal DNA. METHODS: Thirty-seven specimens of A. thylogale collected from N. eugenii (from Parndana, Kangaroo Island, South Australia, and Perup, Western Australia) and S. brachyurus (from Wellington Dam, Western Australia) were characterised using a molecular-phylogenetic approach utilising the first (ITS1) and second (ITS2) internal transcribed spacers. RESULTS: Genetic variation was detected in both ITS1 and ITS2 between specimens of A. thylogale from N. eugenii and S. brachyurus; however, no variation was detected between specimens collected from N. eugenii from Parndana, South Australia, and Perup, Western Australia. Furthermore, the phylogenetic analyses of ITS sequences showed two clades of A. thylogale originating from two hosts, N. eugenii and S. brachyurus, suggesting the presence of cryptic species. CONCLUSIONS: This study provides evidence of genetic variation within A. thylogale based on collections from two different host species. Morphological studies are required to fully confirm the presence of a new species or cryptic species. Further molecular studies using a larger number of specimens are warranted to explore the genetic variation between A. thylogale from different geographical localities.

Understanding Historical Demographic Processes to Inform Contemporary Conservation of an Arid zone Specialist: The Yellow-Footed Rock-Wallaby.

Little genetic research has been undertaken on mammals across the vast expanse of the arid biome in Australia, despite continuing species decline and need for conservation management. Here, we evaluate the contemporary and historical genetic connectivity of the yellow-footed rock-wallaby, Petrogalexanthopusxanthopus, a threatened macropodid which inhabits rocky outcrops across the disconnected mountain range systems of the southern arid biome. We use 17 microsatellite loci together with mitochondrial control region data to determine the genetic diversity of populations and the evolutionary processes shaping contemporary population dynamics on which to base conservation recommendations. Our results indicate the highly fragmented populations have reduced diversity and limited contemporary gene flow, with most populations having been through population bottlenecks. Despite limited contemporary gene flow, the phylogeographic relationships of the mitochondrial control region indicate a lack of structure and suggests greater historical connectivity. This is an emerging outcome for mammals across this arid region. On the basis of our results, we recommend augmentation of populations of P. x.xanthopus, mixing populations from disjunct mountain range systems to reduce the chance of continued diversity loss and inbreeding depression, and therefore maximize the potential for populations to adapt and survive into the future.

Reconstructing the Evolution of Giant Extinct Kangaroos: Comparing the Utility of DNA, Morphology, and Total Evidence.

Combined "total evidence" analysis of molecular and morphological data offers the opportunity to objectively merge fossils into the tree of life, and challenges the primacy of solely DNA based phylogenetic and dating inference, even among modern taxa. To investigate the relative utility of DNA, morphology, and total evidence for evolutionary inference, we sequenced the first near-complete mitochondrial genomes from extinct Australian megafauna: a 40-50 thousand year old giant short-faced kangaroo (Simosthenurus occidentalis) and giant wallaby (Protemnodon anak). We analyzed the ancient DNA and fossil data alongside comparable data from extant species to infer phylogeny, divergence times, and ancestral body mass among macropods (kangaroos and wallabies). Our results confirm a close relationship between Protemnodon and the iconic kangaroo genus complex "Macropus", and unite the giant Simothenurus with the hare-sized Lagostrophus fasciatus (banded hare-wallaby), suggesting that the latter is the closest living link to the once diverse sthenurine kangaroo radiation. We find that large body size evolved multiple times among kangaroos, coincident with expansion of open woodland habitats beginning in the Late Miocene. In addition, our results suggest that morphological data mislead macropod phylogeny reconstruction and in turn can distort total evidence estimation of divergence dates. However, a novel result with potentially broad application is that the accuracy and precision of reconstructing ancestral body mass was improved by tracing body mass on morphological branch lengths. This is likely due to positive allometric correlation between morphological and body size variation-a relationship that may be masked or even misleadingly inverted with the temporal or molecular branch lengths that typically underpin ancestral body size reconstruction. Our study supports complementary roles for DNA and morphology in evolutionary inference, and opens a new window into the evolution of Australia's unique marsupial fauna.

Rapid Pliocene adaptive radiation of modern kangaroos.

Differentiating between ancient and younger, more rapidly evolved clades is important for determining paleoenvironmental drivers of diversification. Australia possesses many aridity-adapted lineages, the origins of which have been closely linked to late Miocene continental aridification. Using dental macrowear and molar crown height measurements, spanning the past 25 million years, we show that the most iconic Australian terrestrial mammals, "true" kangaroos (Macropodini), adaptively radiated in response to mid-Pliocene grassland expansion rather than Miocene aridity. In contrast, low-crowned, short-faced kangaroos radiated into predominantly browsing niches as the late Cenozoic became more arid, contradicting the view that this was an interval of global browser decline. Our results implicate warm-to-cool climatic oscillations as a trigger for adaptive radiation and refute arguments attributing Pleistocene megafaunal extinction to aridity-forced dietary change.

Phylogenetic analysis of the tree-kangaroos (Dendrolagus) reveals multiple divergent lineages within New Guinea.

Amongst the Australasian kangaroos and wallabies (Macropodidae) one anomalous genus, the tree-kangaroos, Dendrolagus, has secondarily returned to arboreality. Modern tree-kangaroos are confined to the wet tropical forests of north Queensland, Australia (2 species) and New Guinea (8 species). Due to their behavior, distribution and habitat most species are poorly known and our understanding of the evolutionary history and systematics of the genus is limited and controversial. We obtained tissue samples from 36 individual Dendrolagus including representatives from 14 of the 17 currently recognised or proposed subspecies and generated DNA sequence data from three mitochondrial (3116 bp) and five nuclear (4097 bp) loci. Phylogenetic analysis of these multi-locus data resolved long-standing questions regarding inter-relationships within Dendrolagus. The presence of a paraphyletic ancestral long-footed and derived monophyletic short-footed group was confirmed. Six major lineages were identified: one in Australia (D. lumholtzi, D. bennettianus) and five in New Guinea (D. inustus, D. ursinus, a Goodfellow's group, D. mbaiso and a Doria's group). Two major episodes of diversification within Dendrolagus were identified: the first during the late Miocene/early Pliocene associated with orogenic processes in New Guinea and the second mostly during the early Pleistocene associated with the intensification of climatic cycling. All sampled subspecies showed high levels of genetic divergence and currently recognized species within both the Doria's and Goodfellow's groups were paraphyletic indicating that adjustments to current taxonomy are warranted.

Molecular and morphological characterisation of Pharyngostrongylus kappa Mawson, 1965 (Nematoda: Strongylida) from Australian macropodid marsupials with the description of a new species, P. patriciae n. sp.

BACKGROUND: Pharyngostrongylus kappa Mawson, 1965 is a nematode (Strongyloidea: Cloacininae), endemic to the sacculated forestomachs of Australian macropodid marsupials (kangaroos and wallaroos). A recent study revealed genetic variation within the internal transcribed spacer region of the nuclear ribosomal DNA among P. kappa specimens collected from Macropus giganteus Shaw and Osphranter robustus (Gould). This study aimed to characterise the genetic and morphological diversity within P. kappa from four macropodid host species, including M. giganteus, O. robustus, O. antilopinus (Gould) and O. bernardus (Rothschild). METHODS: Specimens of P. kappa from M. giganteus and Osphranter spp. from various localities across Australia were examined. The first and second internal transcribed spacers (ITS1 and ITS2, respectively) were amplified using polymerase chain reaction and sequenced. Phylogenetic methods were used to determine the interspecific diversification within P. kappa and its evolutionary relationship with other congeners. RESULTS: Morphological examination revealed that P. kappa from M. giganteus, the type-host, can be distinguished from those in Osphranter spp. by the greater length and number of striations on the buccal capsules. DNA sequences showed that P. kappa from M. giganteus was genetically distinct from that in Osphranter spp., thereby supporting the morphological findings. Based on these finding, a new species from Osphranter spp., Pharyngostrongylus patriciae n. sp., is described. CONCLUSION: Pharyngostrongylus patriciae n. sp. from Osphranter spp. is distinguished from P. kappa based on molecular and morphological evidence. The study highlights the importance of combining molecular and morphological techniques for advancing the nematode taxonomy. Although ITS genetic markers have proven to be effective for molecular prospecting as claimed in previous studies, future utilisation of mitochondrial DNA to validate ITS data could further elucidate the extent of speciation among macropodid nematodes.

Speciation Generates Mosaic Genomes in Kangaroos.

The iconic Australasian kangaroos and wallabies represent a successful marsupial radiation. However, the evolutionary relationship within the two genera, Macropus and Wallabia, is controversial: mitochondrial and nuclear genes, and morphological data have produced conflicting scenarios regarding the phylogenetic relationships, which in turn impact the classification and taxonomy. We sequenced and analyzed the genomes of 11 kangaroos to investigate the evolutionary cause of the observed phylogenetic conflict. A multilocus coalescent analysis using ∼14,900 genome fragments, each 10 kb long, significantly resolved the species relationships between and among the sister-genera Macropus and Wallabia. The phylogenomic approach reconstructed the swamp wallaby (Wallabia) as nested inside Macropus, making this genus paraphyletic. However, the phylogenomic analyses indicate multiple conflicting phylogenetic signals in the swamp wallaby genome. This is interpreted as at least one introgression event between the ancestor of the genus Wallabia and a now extinct ghost lineage outside the genus Macropus. Additional phylogenetic signals must therefore be caused by incomplete lineage sorting and/or introgression, but available statistical methods cannot convincingly disentangle the two processes. In addition, the relationships inside the Macropus subgenus M. (Notamacropus) represent a hard polytomy. Thus, the relationships between tammar, red-necked, agile, and parma wallabies remain unresolvable even with whole-genome data. Even if most methods resolve bifurcating trees from genomic data, hard polytomies, incomplete lineage sorting, and introgression complicate the interpretation of the phylogeny and thus taxonomy.

Resolving kangaroo phylogeny and overcoming retrotransposon ascertainment bias.

Reconstructing phylogeny from retrotransposon insertions is often limited by access to only a single reference genome, whereby support for clades that do not include the reference taxon cannot be directly observed. Here we have developed a new statistical framework that accounts for this ascertainment bias, allowing us to employ phylogenetically powerful retrotransposon markers to explore the radiation of the largest living marsupials, the kangaroos and wallabies of the genera Macropus and Wallabia. An exhaustive in silico screening of the tammar wallaby (Macropus eugenii) reference genome followed by experimental screening revealed 29 phylogenetically informative retrotransposon markers belonging to a family of endogenous retroviruses. We identified robust support for the enigmatic swamp wallaby (Wallabia bicolor) falling within a paraphyletic genus, Macropus. Our statistical approach provides a means to test for incomplete lineage sorting and introgression/hybridization in the presence of the ascertainment bias. Using retrotransposons as "molecular fossils", we reveal one of the most complex patterns of hemiplasy yet identified, during the rapid diversification of kangaroos and wallabies. Ancestral state reconstruction incorporating the new retrotransposon phylogenetic information reveals multiple independent ecological shifts among kangaroos into more open habitats, coinciding with the Pliocene onset of increased aridification in Australia from ~3.6 million years ago.

The complete mitochondrial genome of the eastern grey kangaroo (Macropus giganteus).

We present the complete mitochondrial genome (accession number: LK995454) of an iconic Australian species, the eastern grey kangaroo (Macropus giganteus). The mitogenomic organization is consistent with other marsupials, encoding 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, an origin of light strand replication and a control region or D-loop. No repetitive sequences were detected in the control region. The M. giganteus mitogenome exemplifies a combination of tRNA gene order and structural peculiarities that appear to be unique to marsupials. We present a maximum likelihood phylogeny based on complete mitochondrial protein and RNA coding sequences that confirms the phylogenetic position of the grey kangaroo among macropodids.

Comparative mtDNA analyses of three sympatric macropodids from a conservation area on the Huon Peninsula, Papua New Guinea.

Matschie's tree kangaroo (Dendrolagus matschiei), New Guinea pademelon (Thylogale browni), and small dorcopsis (Dorcopsulus vanheurni) are sympatric macropodid taxa, of conservation concern, that inhabit the Yopno-Urawa-Som (YUS) Conservation Area on the Huon Peninsula, Papua New Guinea. We sequenced three partial mitochondrial DNA (mtDNA) genes from the three taxa to (i) investigate network structure; and (ii) identify conservation units within the YUS Conservation Area. All three taxa displayed a similar pattern in the spatial distribution of their mtDNA haplotypes and the Urawa and Som rivers on the Huon may have acted as a barrier to maternal gene flow. Matschie's tree kangaroo and New Guinea pademelon within the YUS Conservation Area should be managed as single conservation units because mtDNA nucleotides were not fixed for a given geographic area. However, two distinct conservation units were identified for small dorcopsis from the two different mountain ranges within the YUS Conservation Area.

Late pleistocene Australian marsupial DNA clarifies the affinities of extinct megafaunal kangaroos and wallabies.

Understanding the evolution of Australia's extinct marsupial megafauna has been hindered by a relatively incomplete fossil record and convergent or highly specialized morphology, which confound phylogenetic analyses. Further, the harsh Australian climate and early date of most megafaunal extinctions (39-52 ka) means that the vast majority of fossil remains are unsuitable for ancient DNA analyses. Here, we apply cross-species DNA capture to fossils from relatively high latitude, high altitude caves in Tasmania. Using low-stringency hybridization and high-throughput sequencing, we were able to retrieve mitochondrial sequences from two extinct megafaunal macropodid species. The two specimens, Simosthenurus occidentalis (giant short-faced kangaroo) and Protemnodon anak (giant wallaby), have been radiocarbon dated to 46-50 and 40-45 ka, respectively. This is significantly older than any Australian fossil that has previously yielded DNA sequence information. Processing the raw sequence data from these samples posed a bioinformatic challenge due to the poor preservation of DNA. We explored several approaches in order to maximize the signal-to-noise ratio in retained sequencing reads. Our findings demonstrate the critical importance of adopting stringent processing criteria when distant outgroups are used as references for mapping highly fragmented DNA. Based on the most stringent nucleotide data sets (879 bp for S. occidentalis and 2,383 bp for P. anak), total-evidence phylogenetic analyses confirm that macropodids consist of three primary lineages: Sthenurines such as Simosthenurus (extinct short-faced kangaroos), the macropodines (all other wallabies and kangaroos), and the enigmatic living banded hare-wallaby Lagostrophus fasciatus (Lagostrophinae). Protemnodon emerges as a close relative of Macropus (large living kangaroos), a position not supported by recent morphological phylogenetic analyses.

A new species of the basal "kangaroo" Balbaroo and a re-evaluation of stem macropodiform interrelationships.

Exceptionally well-preserved skulls and postcranial elements of a new species of the plesiomorphic stem macropodiform Balbaroo have been recovered from middle Miocene freshwater limestone deposits in the Riversleigh World Heritage Area of northwestern Queensland, Australia. This constitutes the richest intraspecific sample for any currently known basal "kangaroo", and, along with additional material referred to Balbaroo fangaroo, provides new insights into structural variability within the most prolific archaic macropodiform clade--Balbaridae. Qualitative and metric evaluations of taxonomic boundaries demonstrate that the previously distinct species Nambaroo bullockensis is a junior synonym of B. camfieldensis. Furthermore, coupled Maximum Parsimony and Bayesian phylogenetic analyses reveal that our new Balbaroo remains represent the most derived member of the Balbaroo lineage, and are closely related to the middle Miocene B. camfieldensis, which like most named balbarid species is identifiable only from isolated jaws. The postcranial elements of Balbaroo concur with earlier finds of the stratigraphically oldest balbarid skeleton, Nambaroo gillespieae, and suggest that quadrupedal progression was a primary gait mode as opposed to bipedal saltation. All Balbaroo spp. have low-crowned bilophodont molars, which are typical for browsing herbivores inhabiting the densely forested environments envisaged for middle Miocene northeastern Australia.

Studies on the in vitro cultivation of ciliate protozoa from the kangaroo forestomach.

The methods used for culturing rumen protozoa were found to be unsatisfactory for growth of ciliate protozoa from the kangaroo forestomach. Based on published measurements of physical parameters in the marsupial forestomach, several modifications were incorporated into the procedure, i.e., an increase in % hydrogen in the gas phase, adjustment of initial pH of the medium to 6.9-7.0 range, feed only forage as a substrate and incubate at a lower temperature (33-36 °C). Only incubation at the lower temperature increased survival time of the kangaroo protozoa. Two species of Bitricha were still viable after 28 d in culture. Cultures had to be terminated at that time. One of the species differed considerably in size and shape from previously described species and based on 18S rRNA data, may represent a new species of Bitricha. The second species, present in low numbers was identified as Bitricha oblata. In a separate trial, Macropodinium yalanbense survived for 11 d, at which time these cultures also had to be terminated.

Molecular evolution of a novel marsupial S100 protein (S100A19) which is expressed at specific stages of mammary gland and gut development.

S100 proteins are calcium-binding proteins involved in controlling diverse intracellular and extracellular processes such as cell growth, differentiation, and antimicrobial function. We recently identified a S100-like cDNA from the tammar wallaby (Macropus eugenii) stomach. Phylogentic analysis shows wallaby S100A19 forms a new clade with other marsupial and monotreme S100A19, while this group shows similarity to eutherian S100A7 and S100A15 genes. This is also supported by amino acid and domain comparisons. We show S100A19 is developmentally-regulated in the tammar wallaby gut by demonstrating the gene is expressed in the forestomach of young animals at a time when the diet consists of only milk, but is absent in older animals when the diet is supplemented with herbage. During this transition the forestomach phenotype changes from a gastric stomach into a fermentation sac and intestinal flora changes with diet. We also show that S100A19 is expressed in the mammary gland of the tammar wallaby only during specific stages of lactation; the gene is up-regulated during pregnancy and involution and not expressed during the milk production phase of lactation. Comparison of the tammar wallaby S100A19 protein sequence with S100 protein sequences from eutherian, monotreme and other marsupial species suggest the marsupial S100A19 has two functional EF hand domains, and an extended His tail. An evolutionary analysis of S100 family proteins was carried out to gain a better understanding of the relationship between the S100 family member functions. We propose that S100A19 gene/protein is the ancestor of the eutherian S100A7 gene/protein, which has subsequently modified its original function in eutherians. This modified function may have arisen due to differentiation of evolutionary pressures placed on gut and mammary gland developmental during mammal evolution. The highly regulated differential expression patterns of S100A19 in the tammar wallaby suggests that S100A19 may play a role in gut development, which differs between metatherians and eutherians, and/or include a potential antibacterial role in order to establish the correct flora and protect against spiral bacteria in the immature forestomach. In the mammary gland it may protect the tissue from infection at times of vulnerability during the lactation cycle.

Multiple biogeographical barriers identified across the monsoon tropics of northern Australia: phylogeographic analysis of the brachyotis group of rock-wallabies.

The monsoon tropics of northern Australia are a globally significant biodiversity hotspot, but its phylogeography is poorly known. A major challenge for this region is to understand the biogeographical processes that have shaped the distribution and diversity of taxa, without detailed knowledge of past climatic and environmental fluctuations. Although molecular data have great potential to address these questions, only a few species have been examined phylogeographically. Here, we use the widely distributed and abundant short-eared rock-wallaby (Petrogale brachyotis; n = 101), together with the sympatric monjon (P. burbidgei; n = 11) and nabarlek (P. concinna; n = 1), to assess historical evolutionary and biogeographical processes in northern Australia. We sequenced ∼1000 bp of mitochondrial DNA (control region, ND2) and ∼3000 bp of nDNA (BRCA1, ω-globin and two anonymous loci) to investigate phylogeographic structuring and delineate the time-scale of diversification within the region. Our results indicate multiple barriers between the Top End (Northern Territory) and Kimberley (Western Australia), which have caused divergence throughout the Plio-Pleistocene. Eight geographically discrete and genetically distinct lineages within the brachyotis group were identified, five of which are separated by major river valleys (Ord, Victoria, Daly), arid lowlands and discontinuous sandstone ranges. It is likely that these barriers have similarly influenced genetic structure in other monsoonal biota.

Phylogenetic relationships of rock-wallabies, Petrogale (Marsupialia: Macropodidae) and their biogeographic history within Australia.

The rock-wallaby genus Petrogale comprises a group of habitat-specialist macropodids endemic to Australia. Their restriction to rocky outcrops, with infrequent interpopulation dispersal, has been suggested as the cause of their recent and rapid diversification. Molecular phylogenetic relationships within and among species of Petrogale were analysed using mitochondrial (cytochrome oxidase c subunit 1, cytochrome b, NADH dehydrogenase subunit 2) and nuclear (omega-globin intron, breast and ovarian cancer susceptibility gene) sequence data with representatives that encompassed the morphological and chromosomal variation within the genus, including for the first time both Petrogale concinna and Petrogale purpureicollis. Four distinct lineages were identified, (1) the brachyotis group, (2) Petrogale persephone, (3) Petrogalexanthopus and (4) the lateralis-penicillata group. Three of these lineages include taxa with the ancestral karyotype (2n=22). Paraphyletic relationships within the brachyotis group indicate the need for a focused phylogeographic study. There was support for P. purpureicollis being reinstated as a full species and P. concinna being placed within Petrogale rather than in the monotypic genus Peradorcas. Bayesian analyses of divergence times suggest that episodes of diversification commenced in the late Miocene-Pliocene and continued throughout the Pleistocene. Ancestral state reconstructions suggest that Petrogale originated in a mesic environment and dispersed into more arid environments, events that correlate with the timing of radiations in other arid zone vertebrate taxa across Australia.

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Sequencing skippy: the genome sequence of an Australian kangaroo, Macropus eugenii.

Sequencing of the tammar wallaby (Macropus eugenii) reveals insights into genome evolution, and mammalian reproduction and development.

Phylogenetics of the pademelons (Macropodidae: Thylogale) and historical biogeography of the Australo-Papuan region.

Australia and New Guinea share a common biogeographical history and unique vertebrate fauna. Investigation of genetic relationships among the wet forest-restricted pademelons (Macropodidae: Thylogale) provides insight into the historical connections between the two regions and the evolution of the Australasian marsupial fauna. Molecular phylogenetic relationships among Thylogale species were analysed using mitochondrial (12S rRNA and cytochrome b) and nuclear (omega-globin intron) sequence data with Bayesian and maximum likelihood methods. Australian species were resolved as well-supported, monophyletic clades, whereas endemic New Guinean species did not form clades consistent with current morphological taxonomy. Estimates of divergence using a Bayesian relaxed molecular clock model with standard mammalian nucleotide substitution rates indicated radiation of the genus in Australia in the mid to late Miocene. Persistence of Australian species of Thylogale in both southern temperate and northern tropical forests throughout the drying of the Australian continent can be attributed to their having a greater dietary flexibility than other browsing forest macropods. Divergence of the endemic New Guinean lineage occurred in the late Miocene to early Pliocene, indicating the presence of a partially forested landbridge connecting Australia and New Guinea during the Miocene. Mid-Pleistocene divergence between subspecies of the trans-Torresian T. stigmatica implies gene flow during glacial maxima between forest populations in the southern lowlands of New Guinea and the northern Cape York region of Australia. Complex structuring and relatively limited differentiation among populations of the endemic New Guinean species appears to have been influenced by the uplift of land and climate-induced redistribution of forest habitats during the late Pliocene and Pleistocene period. This is in strong contrast to the long evolutionary history and comparatively deep genetic divergence of Thylogale species in Australia. Further evaluation of the species status of the New Guinean Thylogale using more informative nuclear markers and extensive sampling is required.

Family-level relationships among the Australasian marsupial "herbivores" (Diprotodontia: Koala, wombats, kangaroos and possums).

The marsupial order Diprotodontia includes 10 extant families, which occupy all terrestrial habitats across Australia and New Guinea and have evolved remarkable dietary and locomotory diversity. Despite considerable attention, the interrelations of these families have for the most part remained elusive. In this study, we separately model mitochondrial RNA and protein-coding sequences in addition to nuclear protein-coding sequences to provide near-complete resolution of diprotodontian family-level phylogeny. We show that alternative topologies inferred in some previous studies are likely to be artifactual, resulting from branch-length and compositional biases. Subordinal groupings resolved herein include Vombatiformes (wombats and koala) and Phalangerida, which in turn comprises Petauroidea (petaurid gliders and striped, feathertail, ringtail and honey possums) and a clade whose plesiomorphic members possess blade-like premolars (phalangerid possums, kangaroos and their allies and most likely, pygmy possums). The topology resolved reveals ecological niche structuring among diprotodontians that has likely been maintained for more than 40 million years.